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Hepatitis A, B, C, D, E, G,… diagnostic tools and their use . Geert Leroux-Roels Laboratorium voor Klinische Biologie UZ Gent. Human Hepatitis Viruses Discovery and characteristics. Human Hepatitis Viruses Diagnostic tools. Epidemiology of HAV. Hepatitis A virus – diagnostic tools. - PowerPoint PPT Presentation
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Hepatitis A, B, C, D, E, G,…diagnostic tools and their use
Geert Leroux-RoelsLaboratorium voor Klinische Biologie
UZ Gent
Human Hepatitis VirusesDiscovery and characteristics
Virus Discovered Genome Envelope Classification
HAV 1975 ss-RNA Picorna
HBV 1970 ds-DNA HBsAg HepaDNA
HCV 1989 ss-RNA E1-E2 Flaviviridae
HDV 1978 ds-RNA HBsAg viroid
HEV 1990 ss-RNA Caliciviridae
HGV 1995 ss-RNA E1-E2 Flaviviridae
TTV 1997 ss-DNA Circoviridae
Human Hepatitis VirusesDiagnostic tools
Virus Serology Antibodies Antigens
Molecular det/quant
Advanced molec anal
HAV IgM, IgG
HBV HBsAl HBeAl HBcAl
HBsAg HBeAg
det/quant genotype resistance
HCV IgG det/quant genotype
HDV Anti-delta Delta-Ag
HEV IgM, IgG
HGV IgG
TTV IgG det
Epidemiology of HAV
Hepatitis A virus – diagnostic tools• Serology
– Anti-HAV IgM acute HAV infection– Anti-HAV Totaal immunity
- natural- vaccine-induced protective level 10-30 IU/L
• HAV detection in blood and blood productsin faeces, saliva, …in shelfish, food productsin water,sewage,
HAV vaccines• Children (2-15)
Havrix Junior (720 U/dose), 0.5 ml ml/dscheme : 0 and 6-12 mo (2 doses, IM)
• Adults (>15)Havrix 1440 (1440 U/dose, 1 ml/dscheme : 0 and 6-12 mo (2 doses, IM)
Epidemiology of HEVepidemische en sporadische
gevallen
Rare cases in western countries after recent travel in endemic area
Hepatitis E virus
Serology anti-HEV IgM anti-HEV IgG
HEV-RNA
Hepatitis B virus Diagnostic tools and
interpretation
HBV: genome and gene products
3200 baseparen4 open reading frames7 proteins• large (preS1-preS2-S)• Middle (preS2-S)• Small or HBsAg• nucleocapsid or HBcAg• secreted HBe-Ag• Polymerase• X protein
Diagnostic markers of HBV infection
Antigens Antibodies
HBsAg anti-HBsHBcAg anti-HBc-Tot
anti-HBc-IgM
HBeAg anti-HBe
Inflammation and liver cell damageTransaminases ALT/ASTen other biochemical markers
Detection/quantific. of HBV DNACommercial assays- Branched DNA (Bayer)- PCR (Roche Amplicor)In-house nested PCRIn-house real-time PCR
Usefulnessof HBV-DNA quantification
• Diagnosis– Acute HBV infection : HBV DNA is not useful– Chronic HBV infection – Is HBV replicating ?
• HBeAgpos : not useful• HBeAgneg/anti-HBepos : useful, “threshold value” ?
• Prognosis• Therapy
– Decision to treat : ALT, biopsy, HBeAgpos
– Selection treatment– Monitoring : HBV DNA, ALT, HBeAg, anti-HBe,
Evolution of an acute, self-limited HBV infection
Serologic profile of a chronic HBV infection,with a late seroconversion to anti-HBe
Case 1
• 41 years old Afghan male• political refugee• In training for assistant-cook• Medical screening exam for ‘hepatitis’• No symptoms• No history of hepatitis
Results - blood chemistryTest Result Unit Ref. intervalBili Tot 0.66 mg/dL 0.00-1.00Bili Dir 0.10 mg/dL 0.00-0.20Bili Ind 0.55 mg/dL 0.20-0.80ALT 16 U/L 0-41AST 21 U/L 0-38LDH 351 U/L 0-480Alk Fosf 106 U/L 0-128GT 20 U/L 8-61
Results - serology
Test Result
HBsAg Positive
Anti-HBs 46 IU/L
Anti-HBc-Tot Positive
Anti-HAV-IgM Negative
Anti-HCV Negative
Question 1Can HBsAg and anti-HBsoccur concomitantly ?
[HBsAg-anti-HBs] immune complexes
• Are present during the clearance phase of an acute HBV infection in the “window phase” and in some chronic HBV patients
• Routine tests for HBsAg/anti-HBs do not detect immune complexes (IC)
• IC dissociatie (ICD) by treatment of serum (100µl) with HCl (50 µl, 0.5 N, 1h at 37°C) and neutralisation with NaOH (50 µl, 0.5N) (Rabenau et al. 1996)
• Research tests can detect IC’s
HAV serostatus• anti-HAV-IgM antibodies are only
present in the acute phase of HAV infections
• ALT/AST activities are normal• Anti-HAV status (IgG antibodies) would
have been useful to see whether this person still needs HAV vaccination– Food handling– Chronic HBV infection
Results – additional HBV tests
Test Resultaat
HBsAg Positive
Anti-HBs 46 IU/L
Anti-HBc-Tot Positief
HBeAg Negative
Anti-HBe Positive
HBV DNA 8 400 gEq/ml
Question 2Is this person infectious ???
Question 3Does he need treatment ?
• Infectivity is LOW– Spouse and daughter show no signs or
markers of HBV infection (HBsAlneg)– Vaccination of household (sexual contact) !– Twinrix is an alternative for Engerix-B
• Therapy is not indicated– Normal transaminases, low DNA– Follow-up : annually ?
Case 2• Man, born in 1947• 1986 – Ulcerative colitis • 1992 - liver enzymes slightly elevated, no
further investigations • 1998 – abnormal liver tests, alcohol
consumption,• June 1998 – exacerbation of colitis• Nov 2001 - exacerbation of colitis
Evolution of laboratory testsTest May 1998 June 1998 June 1999 Sep 2003
ALT 129 35 24 42AST 94 30 17 33-GT 120 56 28 58HBsAg Pos Pos Pos NegAnti-HBs Neg Neg Neg NegAnti-HBc Pos Pos Pos PosHBeAg Neg NegAnti-HBe Pos Pos HBV DNA <0.7 mEq/ml
Spontaneous seroconversions in chronic HBV
• HBeAg to anti-HBe seroconversion– Inflammation (ALT/AST) = 8-15% per year– Normal ALT <2% in children < 3 years
4-5% in patients > 3 years
• HBsAg to anti-HBs seroconversion– Active HBeAg- hepatitis 0.5% /jaar– Asymptomatic HBeAg- carrier
• In a western population 1-2% /jaar• Post perinatal infection 0.05-0.8% /jaar
HBeAg/anti-HBe seroconversion
• Transition to ‘inactive carrier’– Normalisation of transaminases– Low viral replication and HBV DNA (<105 gEq/ml)
• Active hepatitis with HBeAg-/anti-HBe+
– Elevated transaminases– High(er) HBV DNA (> 105 gEq/ml)– Precore mutation (G1896A: stop codon)– Core/precore promotor mutations
Hepatitis B core and e antigen
-29 aa 183aa 1
HBeAg precursor
HBcAg
aa 1 aa 183
mature HBeAg
aa -10 aa 149 aa 1
AU
G
AU
GP
G18
96A
Interpretation of the serology and its evolution in this patient (case 2)• Spontaneous seroconversion of HBsAg
to anti-HBs• No detectable [HBsAg-anti-HBs] IC’s• HBV DNA detection, quantification and
sequence analysis are needed for a correct diagnosis and prognosis
Case 3
• Man, 45 years• Traffic accident => brain death• Possible organ donor (liver ?)• Serology :• HBsAgneg, anti-HBsneg,anti-HBcpos, anti-
HCVneg,anti-HIVneg, CMVneg
• Biochemistry : no abnormalities
Prevalence of “anti-HBc alone” in low seroprevalence populations
Country (year)
Study group
Population studied
Prevalence number (%)
HBV DNA+ number (%)
Germany 1998
Unselected 18-70 yrs
5300 65 (1.4) 5/65 (7.7)
Germany 1997
IVD users 377 94 (25)
Switserland 1996
Pregnant 9000 104 (1.2) 0/104 (0)
Germany 1998
Blood donors
15,000 27 (0.2) 2/27 (7.4)
Switserland 1999
Blood donors
9751 51 (0.5) 2/51 (3.9)
Prevalence of “anti-HBc alone” in high seroprevalence
populationsCountry (year)
Study group
Population studied
Total HBV(%)
Anti-HBc alone (%)
USA (1984) Male homosexuals
1461 55.2 3.3
USA (1984) Prison inmates
685 18.8 5.0
Senegal (1987)
Africans 3033 88.7 20.8
Hong-Kong (1988)
Chinese 1801 68 11.9
Hong-Kong (1992)
Chinese 4001 40 1.3
Possible causes of “Anti-HBc alone” serology
1
1 = window phase
2
2 = late immunity, only anti-HBc persists
3 = chronic infection with low replication/production of HBV or with HBsAg mutant
3
“Occult hepatitis”
• Typical serology
HBsAgneg, anti-HBsneg, anti-HBcpos
• HBV DNA < detection limit of routine PCR test (e.g. < 200 gEq/mL)
• HBV DNA in the liver
Influence of HCV infection
on HBV replication• HCV core protein suppresses HBV
replication with a factor 2 to 4
• HCV infection reduces the expression of HBsAg in the liver
• Treatment of HCV with IFN also has an effect on HBV
“anti-HBc alone” can be :
1. Window phase2. Late immunity – only sign of past infection3. Chronic infection – “occult infection”4. HBsAg mutant5. “False” positive test result
• really “false positive” – low signal, 2nd EIA
• Core-binding antibodies (IgM vs IgG)
-Nested
PCRLo PCRHi PCRHi PCRHi PCRHBV DNA
+ or -+ or -++--Anti-HBe
--- -++HBeAg
++++++Anti-HBc
+-+++++++HBsAg
== to Hi= to HiHiHi=ALT
HBVReplication
HostImm Resp
LiverDisease
ToleranceActive Hepatitis
HBeAg+ HBeAg-
mutant serocon
Occult Inactivecarrier
Lab Tools IgM anti-HDV HDV-Aganti-HDV HDV-RNA
Hepatitis C virus Diagnostic tools and interpretation
The HCV genome and expressed polyprotein
• Indirect markers – anti-HCV
• ELISA 3-4th generation• Confirmation tests RIBA, LIA
• Direct markers = HCV genome • RT-PCR qualitative and quantitative• Branched-DNA (quantitative) • Genotyping• HCV core antigen
Markers of HCV infection
Anti-HCV assays: generations 1 to 4
1989 1993 2000
Generation 1 2 3 4
Antigens NS3/4 C,NS3,NS4 C,NS3,NS4A/B,NS5A
Sensitivity 95-98% >99%Specificity <95% 99.8%Lag time (wks) 16-24 4-12 4-8
Confirmation of anti-HCV+
• Repeat ELISA on the same sample• Another ELISA on the same sample• Same ELISA on a new sample• Confirmation assays
– RIBA (recombinant immunoblot assay)– LIA (Line immuno assay)
• Confirmation by PCR
Molecular tests for HCV
• Molecular detection – qualitative
• Molecular quantification
• Genotyping
HCV-RNA detection – qualitative
Assay Manufacturer Method Lower limit of detection
Amplicor HCV v2.0
Roche Molec Systems
Manual, qualit PCR
50 IU/ml
Cobas Amplicor HCVV2.0
Roche Molec Systems
Semi-automated, qual PCR
50 IU/ml
Versant HCV RNA qualitative assay
Bayer Corpor, Diagnostics Div
Manual qualit TMA
10 IU/ml
Qualitative HCV-RNA tests
• Confirms diagnosis of HCV infection• Useful for the early diagnosis of acute
hepatitis C• Demonstrates the presence of active
infection• « Gold standard » for documenting
response to therapy
Interpretation of combined anti-HCV and HCV-RNA
resultsAnti-HCV HCV-RNA Interpretation
Neg Neg No infectionEarly post exposure (<1 week)
Neg Pos Acute infection (no Ab’s yet)Chronic in immune deficient p.
Pos Neg Past, recovered infection
Pos Pos Acute HCV infection with Ab’sChronic HCV infection
Treatment of HCV• Clinical studies evaluating the
efficacy of different treatment protocols : drugs, doses, duration, … have revealed the importance of 1) HCV-RNA quantification 2) genotyping
Quantitative HCV RNA tests
• Generally less sensitive than qualitative HCV-RNA test => Taqman !
• Positive in >95% of untreated patients with chronic hepatitis C
• Useful in predicting response to therapy and determination of early virological response (EVR)
Range of HCV-RNA assays
Amplicor Monitor v2.0(PCR)
VersantHCV RNA v3.0(b-DNA)
LCx 25HCV RNA
SuperQuant
20 200 2,000 20,000 200,000 2,000,000 IU/ml
600
615
500,000
7,700,000
2,630,000
30 1,470,000
Molecular Genotyping
• Direct Sequencing– ‘Home-made’ methods: NS5B-, E1-based– 5’-noncoding: Trugene - Visible Genetics
• ‘Reverse Hybridization’– Inno LiPA (Innogenetics)
Analysis of the viral genome sequenceReverse hybridisation of PCR
amplicons
Treatment of HCV
• Interferon-
• Interferon- + Ribavirine
• Pegylated IFN- + Ribavirine
Early stopping rules
• 1997 Consensus conference– IFN monotherapy : stop therapy when
HCV-RNA (sensitive qualitative test) remains POSITIVE after 12 weeks
• 1998 McHutchison – NEJM 339:1485 – IFN + ribavirin : stop therapy when
HCV-RNA (sensitive qualitative test) remains POSITIVE after 24 weeks
n = 390 n = 390 (86%)(86%)
n = 63n = 63(14%)(14%)
2 log reduction or 2 log reduction or HCV RNA (-) HCV RNA (-)
YESYES
NONO
Week 12 (N = 453)Week 12 (N = 453)n = 253n = 253(65%)(65%)SVRSVR
n = 137 n = 137 (35%)(35%)No SVRNo SVR
n = 2n = 2(3%)(3%)
n = 61 n = 61 (97%)(97%)Fried et al. NEJM 2002;347:975-982Fried et al. NEJM 2002;347:975-982
SVRSVR
No SVRNo SVR
PEG-IFN-2a QW + ribavirin QDAll genotypes
2 log drop of HCV-RNA at week 12
• in patients treated with PEG-IFN + ribavirin• undetectable HCV-RNA or log 2 drop at
week 12, is predictive for sustained response (>60%)
• absence of 2 log drop is extremely (>99%) predictive for non-sustained response
• should lead to early stop of treatment• leads to significant cost reduction • avoids inconvenience and side effects
GENOTYPE 2 OR 3GENOTYPE 2 OR 3 GENOTYPE 1 (and 4, 5 or 6)GENOTYPE 1 (and 4, 5 or 6)==
PEG-IFN-PEG-IFN- + 800 mg ribavirin + 800 mg ribavirin24 weeks24 weeks
End-of-treatment virological responseEnd-of-treatment virological responseSustained virological responseSustained virological response
> 2 log decrease> 2 log decreaseor HCV RNA (-)or HCV RNA (-)
at week 12at week 12<2 log decrease<2 log decrease
at week 12at week 12
End-of-treatment virological responseEnd-of-treatment virological responseSustained virological responseSustained virological response
== Stop treatmentStop treatment
or continue in order or continue in order to slow evolutionto slow evolutionof liver diseaseof liver disease
HCV Genotype determinationHCV Genotype determination
Viral load quantification Viral load quantification at baseline and week 12at baseline and week 12
HCV RNA detection HCV RNA detection (sensitive qualitative assay) (sensitive qualitative assay)
at the end of treatment and 24 weeks laterat the end of treatment and 24 weeks later
Liver biopsyLiver biopsy
Bad prognosisBad prognosis==
PEG-IFN-PEG-IFN- + + 1000-1200 mgribavirin1000-1200 mgribavirin
48 weeks48 weeks
Good prognosisGood prognosis==
No treatmentNo treatment
CHRONIC HEPATITIS CCHRONIC HEPATITIS C
HCV RNA detection HCV RNA detection (sensitive qualitative assay) (sensitive qualitative assay)
at the end of treatment and 24 weeks laterat the end of treatment and 24 weeks later
Cost-benefit of monitoring early viral response (EVR) a simulation for Belgium
Cost-benefit of log2 at week 12 Assumptions and data
• Number of new cases per year = 1000• Number of treated cases = 600• Fraction of HCV genotype 1 = 70 %• Cost of quantitative HCV-RNA = 150 Euro per
test, x 2 (w0 and w12) = 300 Euro• Cost of PEG-IFN + Riba = 400 Euro per week• Diagnostic sensitivity (detection of NR) = 33%• NPV of EVR = 100%
Cost-benefit of log2 at week 12
1000 new HCV
600 treated
180 non-gt1 420 gt1
350 EVR
HCV-RNA at w12 63000
HCV-RNA at w0 63000
Cost Benefit
210 SR 140 NSR
70 No EVR
STOP 126000 1008000
w0
w12
W48 End of treatment Net savings 882000
Molecular diagnostic tools for detection and monitoring of HCV
infections• Correct diagnosis• Selection of treatment and duration• Decision to “Stop treatment”
– Reduce costs (medication, doctor visits, ..)– Reduce the discomfort and suffering of patients– Reduce the loss of labour time
– The impact on costs (direct, indirect) will increase if the diagnostic sensitivity of the EVR algorythm can be improved (>33%)
Additional Hepatitis Agents
• 12% o post-transfusion hepatitis unrelated to A-E
• 18% of acute hepatitis unrelated to A-E• Up to 40% of fulminant hepatitis no
etiology is present• Cases of acute hepatitis followed by
aplastic anemia
New viruses not proven to cause human hepatitis
• HGV• TTV• TLMV• TTV-like minivirus• Sanban• Yonban• Sen virus
HGV – GBV-C• Related to HCV - Flaviviridae• Parenteral transmission• Replicates in lymphocytes and not in
hepatocytes• HGV infection prolongs survival in HIV• Does not cause hepatitis and not even
co-morbidity in (frequent) association with HBV or HCV
Prevalence in Belgium Sheng - Doctoral Thesis - KUL 1998
HGV-RNA (%) Anti-E2 Ab (%)
Blood donors 1.8 6
Clotting disord 14.8 25.7
Haemodialysis 17 14.2
Chronic HBV 5.6
Chronic HCV 50.5
Fulminant hep 8.3
TT virus infection in acute and chronic liver diseases and in patients regularly receiving blood products in BelgiumAli S, van Pelt JF, Verslype C, Nevens F, Fevery J, Yap SH – Acta Gastroenterol Belg 2004,67:161
TTV-DNA was present in • 49% of patients with chronic HCV• 54% in patients with chronic HBV• 47% in patients receiving clotting factors• 64% in patients in chronic haemodialysis• 29.7% in (340) healthy blood donors
Analysis of samples for less common forms of hepatitis Dr. Robert Vranckx HEV-AlWetenschappelijk Instituut VolksgezondheidHDV-Ag ?Juliette Wytmansstraat 14 HGV-RNA ?1050 Brussel
Prof. Dr. Patrick Goubau HDV-AlAIDS Reference Laboratory, UCLAvenue Hippocrate 54/92B-1200 Brussels